Thomas Soderling, Ph.D. (biochemistry) - US grants

An investigation will be made of the properties and regulation of glycogen synthase in rabbit muscle and liver in vitro and in vivo. Synthase kinases will be purified and characterized from muscle and liver. Regulation of kinases by calcium, calmodulin, and cyclic nucleotides will be examined. Relationships between phosphorylation, activity, and structure of synthase will be studied. Binding of glucose-6-P and UDP-glucose to muscle synthase will be determined. Phosphorylation of synthase in vivo will be studied in response to epinephrine, insulin, and diabetes. Effects of somatostatin infusion will be examined. The in vivo phosphorylation state of each of the sites in muscle synthase will be determined by analysis of tryptic peptides separated by high performance liquid chromatography.

The objective of these studies is to delineate the molecular mechanism by which the phosphorylation state of skeletal muscle glycogen synthase is altered in diabetes and by insulin treatment. Emphasis will be focused on those protein kinases which phosphorylate sites 2 and 3 in glycogen synthase and/or activate the Mg++/ATP-dependent protein phosphatase I. Effects of the insulin receptor tyrosine protein kinase on these serine/threonine protein kinases and phosphatases will be examined directly using purified enzymes as well as in situ in diaphragm and cultured cells. In the latter case a highly specific antiphosphotyrosine monoclonal antibody affinity column will be utilized to selectively purify protein kinases and phosphatases containing phosphotyrosine. Another approach will be to assess potential effects of insulin-generated "modulator" molecules, derived from a phosphatidylinositol-glycan by activation of a specific phospholipase C, on these glycogen synthase protein kinases and phosphatases. Potential effects of insulin on the subcellular distribution of the protein kinase that activates the Mg /ATP- dependent phosphatase and on the phosphatase itself will be examined.

The objective of these studies is to identify physiological functions of Ca++/ Calmodulin-dependent protein kinase II (CaM- kinase II) in neural tissues. CaM-kinase II is of particular interest in this context since 1) it constitutes about 1% of brain protein, 2) it constitutes about 50% of the postsynaptic density (PSD) protein, and 3) it undergoes a unique autophosphorylation that converts it to a Ca++ -independent form. Our studies will utilize a combination of biochemical and electrophysiological approaches to investigate physiological functions of the cytosolic and membrane-associated brain CaM-kinase II. Potential targets of cytosolic CaM-kinase II that will be investigated include tyrosine hydroxylase, the rate-limiting enzyme in catecholamine and several other regulatory CaM-binding proteins. With reference to tyrosine hydroxylase, our goals will be to establish its in vivo phosphorylation by CaM-kinase II and the regulatory role of an activator protein that is specific for tyrosine hydroxylase that has been phosphorylated by CaM-kinase II. These studies will utilize pinocytotic introduction of antibodies against CaM-kinase II or the activator protein into PC12 cells. A number of known CaM-binding proteins will be screened for specific phosphorylation by the Ca -independent form of CaM-kinase II. We are especially interested in regulatory phosphorylation sites which are blocked when Ca /CaM is bound to the CaM-binding proteins. A major focus of these studies will be the CaM-kinase II localized in the PSD. Of special interest is the potential role of this kinase in regulating certain ion channels, specifically, the NMDA- receptor/ion channel and the dihydropyridine-sensitive Ca channel. These studies will combine radioligand binding analyses and patch- clamp studies. We are particularly interested in potential regulation of these ion channels by protein phosphorylation and by GTP-binding proteins. The NMDA channel is of special relevance due to its likely involvement in synaptic plasticity such as long-term potentiation and kindling, and perhaps in epilepsy.

The objective of these studies is to delineate the molecular mechanisms by which brain Ca++/calmodulin(Cam)-dependent protein kinase II (CaM-kinase II) is regulated by binding of Ca /CaM and by autophosphorylation, both with the purified kinase and in intact cells and tissues. Long-term regulation of brain CaM-kinase II by covalent mechanisms (i.e., autophosphorylation) is of special interest because of the putative neuronal functions of this kinase. There is good evidence that presynaptically CaM-kinase II is intimately involved in Ca-dependent regulation of catecholamine biosynthesis and neurotransmitter exocytosis. CaM-kinase II comprises 50% of protein in the postsynaptic density of excitatory synapses and may be involved in long-term alterations of synaptic plasticity. Previous studies from this and other laboratories have led to the formulation of a model for the regulation of CaM-kinase II by binding of Ca/CaM and by autophosphorylation. This model will be tested using synthetic peptides, derived from the known sequence of the 50 kDa subunit of the brain CaM-kinase II, to characterize CaM-binding and substrate-directed inhibitory domains within the kinase sequence. Amino acids essential to these functions will be identified using altered peptide sequences. The effects of phosphorylation of consensus phosphorylation sites flanking these two domains will be assessed in terms of the function of these domains. These results will be utilized in the design of in vitro site-specific mutagenesis to test the functions of specific amino acids on the properties of the kinase. Lastly the ability of CaM- kinase II to undergo autophosphorylation and formation of Ca independent species in intact cells will be assessed in hippocampal brain slices and in cultured hippocampal cells and PC12 cells in response to Ca influx. These studies will further our understanding of the regulation and function of this multifunctional Ca++ -dependent kinase.

The objective of these studies is to delineate the molecular mechanism by which the phosphorylation state of skeletal muscle glycogen synthase is altered in diabetes and by insulin treatment. Emphasis will be focused on those protein kinases which phosphorylate sites 2 and 3 in glycogen synthase and/or activate the Mg++/ATP-dependent protein phosphatase I. Effects of the insulin receptor tyrosine protein kinase on these serine/threonine protein kinases and phosphatases will be examined directly using purified enzymes as well as in situ in diaphragm and cultured cells. In the latter case a highly specific antiphosphotyrosine monoclonal antibody affinity column will be utilized to selectively purify protein kinases and phosphatases containing phosphotyrosine. Another approach will be to assess potential effects of insulin-generated "modulator" molecules, derived from a phosphatidylinositol-glycan by activation of a specific phospholipase C, on these glycogen synthase protein kinases and phosphatases. Potential effects of insulin on the subcellular distribution of the protein kinase that activates the Mg /ATP- dependent phosphatase and on the phosphatase itself will be examined.

The objective of these studies is to identify physiological functions of Ca++/ Calmodulin-dependent protein kinase II (CaM- kinase II) in neural tissues. CaM-kinase II is of particular interest in this context since 1) it constitutes about 1% of brain protein, 2) it constitutes about 50% of the postsynaptic density (PSD) protein, and 3) it undergoes a unique autophosphorylation that converts it to a Ca++ -independent form. Our studies will utilize a combination of biochemical and electrophysiological approaches to investigate physiological functions of the cytosolic and membrane-associated brain CaM-kinase II. Potential targets of cytosolic CaM-kinase II that will be investigated include tyrosine hydroxylase, the rate-limiting enzyme in catecholamine and several other regulatory CaM-binding proteins. With reference to tyrosine hydroxylase, our goals will be to establish its in vivo phosphorylation by CaM-kinase II and the regulatory role of an activator protein that is specific for tyrosine hydroxylase that has been phosphorylated by CaM-kinase II. These studies will utilize pinocytotic introduction of antibodies against CaM-kinase II or the activator protein into PC12 cells. A number of known CaM-binding proteins will be screened for specific phosphorylation by the Ca -independent form of CaM-kinase II. We are especially interested in regulatory phosphorylation sites which are blocked when Ca /CaM is bound to the CaM-binding proteins. A major focus of these studies will be the CaM-kinase II localized in the PSD. Of special interest is the potential role of this kinase in regulating certain ion channels, specifically, the NMDA- receptor/ion channel and the dihydropyridine-sensitive Ca channel. These studies will combine radioligand binding analyses and patch- clamp studies. We are particularly interested in potential regulation of these ion channels by protein phosphorylation and by GTP-binding proteins. The NMDA channel is of special relevance due to its likely involvement in synaptic plasticity such as long-term potentiation and kindling, and perhaps in epilepsy.

The objective of these studies is to delineate the molecular mechanisms by which brain Ca++/calmodulin(Cam)-dependent protein kinase II (CaM-kinase II) is regulated by binding of Ca /CaM and by autophosphorylation, both with the purified kinase and in intact cells and tissues. Long-term regulation of brain CaM-kinase II by covalent mechanisms (i.e., autophosphorylation) is of special interest because of the putative neuronal functions of this kinase. There is good evidence that presynaptically CaM-kinase II is intimately involved in Ca-dependent regulation of catecholamine biosynthesis and neurotransmitter exocytosis. CaM-kinase II comprises 50% of protein in the postsynaptic density of excitatory synapses and may be involved in long-term alterations of synaptic plasticity. Previous studies from this and other laboratories have led to the formulation of a model for the regulation of CaM-kinase II by binding of Ca/CaM and by autophosphorylation. This model will be tested using synthetic peptides, derived from the known sequence of the 50 kDa subunit of the brain CaM-kinase II, to characterize CaM-binding and substrate-directed inhibitory domains within the kinase sequence. Amino acids essential to these functions will be identified using altered peptide sequences. The effects of phosphorylation of consensus phosphorylation sites flanking these two domains will be assessed in terms of the function of these domains. These results will be utilized in the design of in vitro site-specific mutagenesis to test the functions of specific amino acids on the properties of the kinase. Lastly the ability of CaM- kinase II to undergo autophosphorylation and formation of Ca independent species in intact cells will be assessed in hippocampal brain slices and in cultured hippocampal cells and PC12 cells in response to Ca influx. These studies will further our understanding of the regulation and function of this multifunctional Ca++ -dependent kinase.

Calcium/calmodulin (Ca2+/CaM) dependent protein kinases (CaM-kinases) are important signal transduction enzymes which are particularly abundant in brain. In brain and other tissues they are crucial in regulating numerous physiological functions including neurotransmitter synthesis and release, several ion channels and gene expression. This proposal will examine important structure/function and regulatory features of two CaM-dependent kinases, CaM-kinase II and CaM-kinase IV. These studies will utilize purified enzymes expressed in the baculovirus/Sf9 cell system. Techniques of protein chemistry, site-specific mutagenesis and enzymology will be employed. Major components of these studies will include: 1. Detailed analysis of the functionality of the autoinhibitory domain of CaM-kinase II, its regulation by autophosphorylation, and its interaction with the catalytic domain. These results will be used as a biochemical basis for molecular modeling of CaM-kinase II. These studies will clarify the regulatory model for this enzyme which has important consequences for its involvement as a long-term sensor of synaptic activity. 2. The structure and regulation of CaM-kinase IV and its substrate specificity will be determined. Particular emphasis will be placed on its enzymatic properties which may be important for Ca2+ -dependent transcriptional regulation of immediate early genes. 3. Attempts will be made to obtain crystals of CaM-kinase II which would be suitable for subsequent X-ray determination of higher ordered structure. These studies will further our understanding of the structural, regulatory and enzymatic properties of these signal transduction enzymes, thereby furthering our understanding of their probable physiological roles in learning and memory, epilepsy and stroke.

Excitatory neurotransmission in the central nervous system is mediated largely by the neurotransmitter glutamate acting on specific glutamate receptors. It is well known that the efficiency of transmission at these glutamatergic synapses can be decreased (long-term depression) or enhanced (long-term potentiation) for prolonged periods of time. This synaptic plasticity is due to both alterations in the release of neurotransmitter (presynaptic mechanism) and in the responsiveness of the postsynaptic neuron. This grant application will investigate several Ca2+ dependent mechanisms that may account for postsynaptic changes in glutamate receptor ion channels (GluRs). Results from our laboratory, as well as from several other research groups, has provided strong evidence that a key player in these postsynaptic Ca2+ dependent changes is phosphorylation of glutamate receptor ion channels (GluRs) by Ca2+/calmodulin-dependent protein kinase II (CaM-K II) and perhaps dephosphorylation by calcineurin. This grant application will investigate regulatory functions of CaM-K II and calcineurin on all three types of GluRs. The specific aims are: l. Investigate the phosphorylation and regulation of AMPA-type GluRs by CaM-K II and protein phosphatases. 2. Investigate the regulation of kainate-type GluRs by CaM-K II. 3. Determine the roles of protein kinases and phosphatases in two Ca2+ dependent modulations of NMDA-type GluRs. These studies will include both biochemical, molecular biological and electrophysiological techniques using cultured hippocampal neurons and the various GluRs expressed in HEK 293 cells. The results of these studies have strong implications for modulating synaptic efficiency in paradigms of learning and memory and for neuronal excitability in epilepsy.

The overall mission of this core facility is to provide the necessary equipment and technical suppport for tissue culture and baculovirus services to allow rigorous pursuit of the goals of the Program project. The rationale is to establish these services in a central location (third floor, Vollum Institute) to be shared by members of the four research groups, in order to further scientific exchange between the participating laboratories and to eliminate duplication in costs of equipment, supplies and salaries. Establishment of this core facility will therefore save a substantial amount of research effort and costs for each of the component laboratories. The core facility will provide several services critical for the development of each project.

Elevated intracellular calcium is one of the most important signaling molecules in cells, especially in neural tissues. One of the important actions of calcium is to regulate the transcription of selected genes, thereby altering the phenotype of the cell. Studies from a number of laboratories, including our own, have demonstrated that a family of calmodulin-dependent protein kinases mediate many of these Ca2+- dependent transcriptional events. In particular, we have focused on CaM-kinase IV which can phosphorylate several transactivating proteins such as CREB and SRF. In the past two years we have partially characterized a CaM-kinase cascade consisting of a CaM- kinase kinase which can phosphorylate and activate CaM-kinase IV and CaM-kinase I. In the current grant application we will further characterize the involvement of this CaM-kinase cascade by: 1) identifying additional upstream components of the cascade (e.g. CaM- kinase kinase kinase) or anchoring proteins for CaM-kinase kinase. 2) identifying additional transactivating proteins for CaM-kinase IV by examining transcriptional regulation of the NGFI-B gene by the CaM- kinase cascade. 3) determining the sites and physiological relevance of cross-talk between the CaM-kinase cascade and the MAP-kinase cascade. These studies will be performed in a variety of cultured cells, especially PC12 cells, and will utilize biochemical and molecular biological techniques. The results of this study have implications for modulating cellular apoptosis and synaptic plasticity due to Ca2+- dependent gene transcription.

DESCRIPTION: (Adapted from the applicant's abstract): Calcium/calmodulin-dependent protein kinases (CaM-kinases) are important signal transduction enzymes which are particularly abundant in brain. These kinases are crucial for regulating numerous Ca2+-triggered physiological functions including hormone actions, neurotransmitter synthesis and release, modulating several ion channels, mediating aspects of learning and memory, and triggering selective transcription of key genes. This proposal will examine important structural and regulatory properties of three CaM-kinases: CaM-kinase II, CaM-kinase IV and CaM-kinase kinase. These studies will utilize purified enzymes in vitro as well as studies on mammalian cells transfected with wild- type and mutants of these kinases. Techniques of protein chemistry, site-specific mutagenesis and enzymology will be employed. Major components of these studies will include: 1. Cloning and characterization of a protein inhibitor of CaM-KII. The biochemical mechanism(s) regulating its interaction with and inhibition of CaM-KII will be determined in vitro and in cultured cells. 2. We will study mechanisms (e.g., nuclear localization signals) involved in regulating the distribution of CaM-KIV between the nucleus, where it regulates gene transcription, and the cytosol, where it exerts cross-talk with the MAP-kinase pathways. We will also determine the biochemical mechanism by which Ca2+independent activity is generated in CaM-kinase IV upon its activation by CaM-kinase kinase. 3. Extensive studies will be performed on structure/function aspects of the a and b isoforms of CaM-kinase kinase. In particular, regulatory functions of unique sequences in these proteins will be examined in terms of their potential autoregulatory properties or interactions with other cellular proteins. Activation by hormones and neurotransmitters will be assessed. These studies will further our understanding of the enzymatic and regulatory properties of these key signaling proteins, thereby furthering our understanding of their roles in hormone actions, learning and memory, epilepsy and stroke.

DESCRIPTION (provided by applicant): Calcium is a ubiquitous signaling ion, especially in excitable cells such as neurons. Although there are numerous receptor proteins for Ca2+, the most prominent is calmodulin (CaM). The Ca2+/CaM complex can modulate the functionality of numerous proteins in the cells including several Ser/Thr protein kinases. The Ca2v/CaM-dependent protein kinase (CaM-K) cascade is made up of CaM-kinase I (CaM-KI), CaM-kinase IV (CaM-KIV) and their upstream activating kinase CaM-kinase kinase (CaM-KK). These CaM-Ks are particularly abundant in neurons and are important in diverse cellular functions such as gene transcription, microtubule assembly, apoptosis, and synaptic plasticity. The CaM-K cascade also exhibits regulatory cross-talk with other signaling pathways such as the cAMP and MAP-kinases. The investigations proposed in this application will focus on CaM-KK. These studies will utilize purified, recombinant enzymes as well as endogenous or transfected CaM-KK mutants in cultured cells.Major components of these studies will include: 1. Investigating the regulation of CaM-KK by phosphorylation/dephosphorylation in a variety of cells. CaM-KK activity can be modulated in vitro by PKA and CaM-KI through phosphorylation of identified sites, and preliminary evidence indicates dynamic changes in cultured cells. We will extend these studies using cultured cells stimulated by agonists. Attempts will be made to identify the relevant kinases and phosphatases using selective inhibitors. 2. Web based motif programs predict that CaM-KK can interact with several scaffold or bridging proteins, and these predictions are supported by our preliminary studies. Motifs in CaM-KK required for these interactions will be determined, and the effects of agonist treatments of cultured cells on these interactions will be determined. 3. CaM-KK may be localized in the cell due to its interactions with these proteins. We are particularly interested in the dynamic localization of CaM-KK in growth cones, where it may modulate neurite extension, and the cell nucleus, where it regulates gene transcription through CaM-KIV.These studies will further our understanding of the functions of CaM-KK in cellular Ca2+ signaling and their roles in neurotransmitter/hormone responses, neuronal development and cell death, and synaptic plasticity.

Intracellular calcium (Ca2+) is one of the most important signaling molecules in cells, especially in neural tissues. Most effects of Ca2+ are transduced through specific Ca2+ binding proteins such as calmodulin (CaM) which interact with many proteins including a family of CaM- kinases (CaM-Ks). My laboratory has characterized several members of this CaM-K family in terms of molecular properties and regulation. More recently we have begin to investigate physiological functions for these CaM-Ks and identify their substrates. In this grant we will focus on the CaM-K cascade which is comprised of CaM-KK and its downstream targets CaM-KI, CaM-KI, CaM-KIV and protein kinase B (PKB). We will study the role of the CaM-K cascade in two important developmental aspects, apoptosis and neurite outgrowth, using cerebellar granule neurons. Aim 1. Role of CaM-K cascade in inhibiting apoptosis. A variety of dominant-interfering and constitutively active mutant of KK, KIV, PKB, CREB and CBP will be used to explore their roles in regulating apoptosis in granule cells maintained in either HK (25 mM) or LK (5 mM kci). Attempts will be made to identify regulated genes, such as those encoding BDNF or Bcl-2 family members. Aim 2. Role of CaM-K cascade in promoting neurite outgrowth. The same reagents as in Aim 1 will be employed to determine their effects on neurite outgrowth in granule neurons maintained in HK. Attempts will be to make to determine whether the CaM-K cascade is acting through nuclear events or at the dendrites and growth cone and to identify possible substrates of KI. These studies will further our understanding of the molecular pathways regulating these important developmental functions in neurons. They will also contribute to our knowledge of the physiological functions of the CaM-K cascade.

[unreadable] DESCRIPTION (provided by applicant): In the mammalian CNS rapid excitatory neurotransmission is mediated largely by glutamate acting on synaptic ionotropic AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs). At CA1 synapses in the hippocampus the strength of this transmission can be regulated by different patterns of neuronal activity. This bidirectional synaptic plasticity is a cellular mechanism for learning and memory. Long-term potentiation (LTP), a model of synaptic plasticity, is mediated in part by Ca2+ activation of CaMKII which phosphorylates several postsynaptic proteins, including AMPARs, to acutely regulate their function. However, molecular mechanisms responsible for trafficking of AMPARs into synapses, a crucial mechanism for LTP, and perhaps changes in their subunit composition to favor Ca2+-permeable AMPARs (CP- AMPARs) are poorly understood. We have preliminary evidence that another class of CaMKs, namely CaM- kinase kinase (CaMKK) and its downstream target CaMKI, may be involved in synaptic trafficking of CP- AMPARs. Furthermore, our studies on developmental formation of spines and synapses in cultured neurons have identified a multiprotein signaling complex that enhances CaMKK/CaMKI phosphorylation of PIX, a Rac guanine-nucleotide exchange factor that promotes spinogenesis through regulation of the actin cytoskeleton. Based on preliminary results, we propose that CaMKI may also be involved in regulating spine density and morphology during structural plasticity that occurs during LTP maintenance and that CP-AMPARs are essential for these structural changes. The main goal of this application is to identify novel signaling pathways involving CaMKK and CaMKI that mediate AMPAR trafficking/recomposition and structural plasticity during synaptic potentiation, probably through modulating the actin cytoskeleton. However, we will also look at possible involvement of CaMKII in these events. This will be a multifaceted investigation that will utilize 1) cultured hippocampal neurons, acute and organotypic cultured hippocampal slices, 2) induction of synaptic potentiation by theta-burst stimulation or treatment with the NMDA receptor co-agonist glycine, 3) pharmacological reagents, transfected dominant-negative and constitutively-active constructs and siRNAs, and 4) electrophysiological and biochemical analyses. Our laboratory has extensive experience with all these approaches and we are in a unique position to undertake this investigation. The cumulative results from these studies will further our understanding of molecular mechanisms underlying synaptic potentiation during paradigms of learning and memory. Furthermore, our studies on signaling pathways that regulate spine morphology and density will have strong clinical implications as several forms of mental retardation (e.g., Down's, Rett, Fragile X and fetal alcohol syndromes) are associated with aberrant spine structures and/or numbers. PUBLIC HEALTH RELEVANCE: These studies will elucidate molecular mechanisms that contribute to synaptic plasticity, a cellular model of learning and memory in the brain. Furthermore, they will identify signaling pathways responsible for formation of calcium-permeable AMPA-type glutamate receptors that are promote cell death in several neuropathologies such as ischemia, stroke, and Alzheimer's disease. Lastly, we will investigate mechanisms for formation of functional dendritic spines, which are abnormal in several forms of mental retardation (e.g., Fragile X, Rett and Down's syndromes). [unreadable] [unreadable]

Intracellular calcium is a critical signaling molecule in the brain for many functions including neuronal development and synaptic plasticity. A family of Ca2+/calmodulin-regulated protein kinases (CaMKs) are key regulators of several of these events. During the past several years we identified regulatory functions of CaM-KK and its downstream effector CaMKI in the morphology and motility of axonal growth cones and basal axonal outgrowth, activity-dependent dendritic arborization and early-phase long-term potentiation. In the current application we will focus on two critical aspects of neuronal development, axon specification and formation of dendritic spines, for which we have preliminary evidence for involvement of CaMKK and CaMKI. We will analyze the activation state of CaMKK and CaMKI isoforms in different subcellular compartments of cultured hippocampal neurons using several approaches including FRET analysis. From proteomic analysis, we have evidence for the existence of a CaMKK signalsome containing beta-PIX and GIT1, and FIX is an in vitro substrate for CaMKI. The role of this signaling complex, as well as several other CaMKI substrates (Numb and stathmin 2) known to regulate the actin and microtubule cytoskelton, in axon specification and spinogenesis will be explored. Phosphorylation of these proteins by CaMKI in neurons will be studied using phospho-specific antibodies. These signaling pathways from CaMKI to axonogensis and spinogenesis will be defined using multiple, independent approaches including dominant-interferring and constitutively-active constructs, pharmacological inhibitors, and RNA interference. The primary experimental system will be cultured hippocampal neurons, but results will be extended into cultured hippocampal slices where possible. Identifying the fundamental signaling pathwaysthat modulate these essential neuronal functions is foundational to understanding normal and pathological brain functions including axon regeneration and mental retardation. Structural abnormalities in dendritic arborization and spine formation, both in human patients and in mouse models, are common to numberous forms of mental retardation including Fragile X, fetal alcohol, Down's and Rett syndromes as well as several evironmental causes of retardation. Thus, these studies have strong implications for underlying causes of mental diseases.